Method for enriching and isolating bacteria which aerobically degrades PCNB

ABSTRACT

It is an object of the invention to improve a conventional soil percolation technique to thereby provide a method of enriching and isolating decomposing bacteria decomposing an organochlorine agricultural chemical PCNB which is difficult to decompose, in a short time period, and to provide decomposing bacteria for efficiently processing PCNB. To practice the method, an enrichment soil layer  2  is formed by mixing a soil containing an organochlorine agricultural chemical PCNB with a fragmented porous material having an infinite number of micropores and at the same time a greater adsorptivity for adsorbing PCNB than the soil, and an inorganic salt medium  3  containing a carbon and nitrogen source, the carbon and nitrogen source being formed by only PCNB, is circulated through the enrichment soil layer  2 , thereby enriching the aerobic bacteria  Burkholderia cepacia  in the fragmented porous material.

RELATED APPLICATIONS

This application is a national stage application under 35 USC 371 andclaims priority from PCT/JP99/02420, filed May 11, 1999, which claimspriority from Japanese Patent Application No. 135156/1998, filed May 18,1998. This application is also related to Japanese Patent ApplicationNo. Hei 9-30176. All of these applications are incorporated herein byreference.

TECHNICAL FIELD

This invention relates to a technique of enriching decomposing bacteriawhich can be used to treat soil polluted by organochlorine compounds,such as agricultural chemicals, particularly to prevent contamination ofriver water and groundwater caused by agricultural chemicals in soil,and a technique of isolating the decomposing bacteria by utilizing theenriching technique, and more particularly to techniques of these kindsfor enriching and isolating decomposing bacteria for decomposingorganochlorine agricultural chemicals difficult to decompose.

BACKGROUND ART

To maintain today's agricultural production, agricultural chemicalscannot be dispensed with, and to conserve flora in golf courses or thelike as well, agricultural chemicals are used in large quantities. Onthe other hand, there is a concern that agricultural chemicals work ascontaminants to have undesirable effects on the environment, especiallyto be a pollution source of river water and groundwater. Anorganochlorine agricultural chemical pentachloronitrobenzene (“PCNB”)used for killing bacteria causing soil disease is pointed out to be oneof contaminants causing such a pollution. PCNB is an organochlorinecompound very difficult to decompose, and as matters stand, a method ofefficiently disposing PCNB remaining in soil and the like has not beenproposed.

To restore soil contaminated by organochlorine compounds, such asagricultural chemicals, with decomposing bacteria has been considered tobe a useful technique. This technique makes use of decomposing bacteria,among microbes inhabiting in soil in enormous numbers, which are capableof decomposing organic compounds serving as functional skeletons inagricultural chemicals and the like, thereby rendering the organiccompounds harmless or eliminating the same from the environment.Therefore, it is possible to eliminate contaminants, such asagricultural chemicals or the like, from the environment by collectingbacteria which are capable of decomposing organochlorine compounds andexploiting such capabilities of the decomposing bacteria.

The method of selective enrichment/isolation of a specific kind ofbacteria from various soil-inhabiting bacteria includes a soilpercolation technique in which a column or the like is filled with soilcontaining inhabiting decomposing bacteria to form an enrichment soillayer, and an inorganic salt medium, which contains only organochlorinecompounds, such as agricultural chemicals, as solo carbon and nitrogensources, is continuously circulated through the enrichment soil layer,whereby a specific kind of decomposing bacteria, that is, decomposingbacteria which is capable of using the carbon or nitrogen sourcecontained in the inorganic salt medium for assimilation or co-metabolismis selectively enriched for isolation. Actually, however, the aboveconventional soil percolation technique generally takes a long timeperiod of one half to one year to enrich and isolate decomposingbacteria, and depending on the kind of an organic compound, there arecases where no suitable decomposing bacteria can be enriched by themethod. The fact that such a long time period is required is a largeimpediment encountered in putting to practical use the river water orgroundwater pollution control technique using decomposing bacteria.

The present invention has been made under these circumstances, and anobject thereof is to improve the conventional soil percolation techniqueto thereby provide a method of enriching and isolating decomposingbacteria capable of decomposing an organochlorine agricultural chemicalPCNB which is difficult to decompose, in a short time period, and toprovide decomposing bacteria for efficiently disposing of PCNB.

DISCLOSURE OF THE INVENTION

To attain the object, the present inventors have improved theconventional soil percolation technique in the following respects, andthereby established the technique which is capable of largely enhancingthe speeds of enrichment and isolation of decomposing bacteria. The gistof the improvement is that a porous material having an infinite numberof micropores is fragmented to pieces of approximately several mm to tenand several mm in size such that the porous material can be handled withease and at the same time has a large effective surface area, and thenthe fragmented porous material is mixed into an enrichment soil layer asan artificial microhabitat. According to this technique, decomposingbacteria can be effectively enriched and isolated over a time period ofapproximately three weeks to three months, although the required timeperiod is slightly different depending on the kind of a contaminant andthe kind of bacteria decomposing the contaminant. The inventors havealready filed a patent application concerning the technique (JapanesePatent Application No. Hei 9-30176).

According to the present invention, by using the improved soilpercolation technique, a specific kind of decomposing bacteria wereselectively enriched and isolated by continuously circulating aninorganic salt medium containing an organochlorine agricultural chemicalPCNB as only sources of carbon and nitrogen to the enrichment soillayer. As a result, aerobic bacteria could be obtained in a very shorttime period which effectively carries out complete decomposition ofPCNB. Out of the enriched and isolated decomposing bacteria, threestrains which have a high PCNB-decomposing activity were examined foridentification, and it was found that the three strains belong toaerobic bacteria named Burkholderia cepacia. As far as the inventorsknow, decomposing bacteria for decomposing PCNB are mostly anaerobicbacteria, and Burkholderia cepacia obtained by the improved soilpercolation technique (Burkholderia cepacia KTYY97, National Instituteof Bioscience and Human Technology Agency of Industrial Science andTechnology, 1-3, Higashi 1-chrome, Tsukuba-shi, Ibaraki 305-8566, Japan,Receipt No. FERM BP-6721, Received May 18, 1998, hereinafter referred toas “the present decomposing bacteria”) provides possibility of quitenovel uses since this bacteria are aerobic and completely decomposes ordegrades PCNB.

It is found that if a loopful—using an inoculating needle—of the presentdecomposing bacteria is added to 30 ml of an inorganic salt mediumcontaining PCNB in a concentration of 3 to 4 mg/liter, it is possible tocompletely decompose PCNB in approximately five days such that allchlorine atoms bonded to each PCNB molecule are removed from themolecule. Further, the study of the present inventors revealed that thepresent decomposing bacteria are capable of decomposing even anorganochlorine agricultural chemical CNP which has been conventionallyconsidered to be very difficult to decompose. Aerobic bacteria, such asthe present decomposing bacteria, are less restricted in the manner ofhandling the same, unlike anaerobic bacteria, and hence only by applyingthe present decomposing bacteria to a soil contaminated by PCNB, it ispossible to effectively decompose the PCNB, which makes it possible toprevent river water and groundwater pollution.

The present decomposing bacteria can be enriched and isolated by theimproved soil percolation technique proposed by the present inventors.This method comprises mixing a soil containing an organochlorineagricultural chemical PCNB with a fragmented porous material having aninfinite number of micropores and at the same time a greateradsorptivity for adsorbing PCNB than the soil to form an enrichment soillayer, and circulating through the enrichment soil layer an inorganicsalt medium containing a carbon and nitrogen source, the carbon andnitrogen source being formed by only PCNB, thereby enriching thedecomposing bacteria in the fragmented porous material.

The soil containing the organochlorine agricultural chemical PCNB ispreferably a soil continuously using PCNB as an agricultural chemical.The present decomposing bacteria are hardly populated in ordinary soils,and on the other hand, is relatively thickly populated in a soil usingPCNB. Therefore, by making use of such a soil, the present decomposingbacteria can be efficiently enriched.

It is preferred that the fragmented porous material used for theenrichment is formed by fragmenting a porous material having an infinitenumber of micropores, such as charcoal, to pieces of several to ten andseveral mm in size, and the fragmented porous material is mixed as anartificial microhabitat into the enrichment soil layer. Further, tocarry out speedy enrichment of the present decomposing bacteria, it ispreferable to use charcoal of broad-leaved tree which is baked at a lowor medium temperature of approximately 400 to 700° C., with a volumeratio of micropores with a diameter of approximately 5 to 20 μm to atotal of micropores being 10% or more, the inside of micropores beingslightly alkaline with pH of approximately 7 to 8, and further with aspecific surface area of the material being approximately 80 to 120m²/g. The results of experiments of enrichment made by using variousartificial microhabitats different in micropore distribution, pH withinmicropores, and specific surface area showed that the artificialmicrohabitat satisfying the above conditions makes it possible to enrichthe present decomposing bacteria at a highest speed and with a highestefficiency. Presumably, this is because the artificial microhabitatsatisfying the above conditions is readily inhabited by the presentdecomposing bacteria and a manner of adsorption of PCNB in themicropores permits the present decomposing bacteria to use PCNBefficiently.

The method of isolating the present decomposing bacteria is carried outby using the above enriching method, and comprises mixing a fragmentedporous material having the present decomposing bacteria enriched thereininto a new fragmented porous material to form an enrichment layerconsisting of the fragmented porous materials only, and circulatingthrough the enrichment layer an inorganic salt medium containing acarbon and nitrogen source, the carbon source and the nitrogen sourcebeing formed by only PCNB, thereby effecting enrichment of the presentdecomposing bacteria in the new fragmented porous material as well toincrease a degree of enrichment of the present decomposing bacteria, forisolation thereof. To carry out this isolating operation at a high speedand with efficiency, it is preferred that the artificial microhabitatsatisfying the above conditions be used as the new fragmented porousmaterial and the isolating operation be repeatedly carried out.

Further, the fragmented porous material having the present decomposingbacteria enriched therein by the enriching and isolating methodsaccording to the invention can be used as an organochlorine agriculturalchemical-decomposing bacteria holdback carrier. Thus, the capability oftaking out the present decomposing bacteria in the form of amicrohabitat which can be easily handled, i.e. as a carrier holding thepresent decomposing bacteria facilitates application of the holdbackcarrier holding the present decomposing bacteria to a soil contaminatedby PCNB, e.g. mixing the same into a target soil where PCNB should bedisposed of, and makes it possible to decompose PCNB in the soil withoutproviding a special facility.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing a structural formula of PCNB;

FIG. 2 shows a table showing physical properties of each artificialmicrohabitat used in the present embodiment;

FIG. 3 is a diagram schematically showing a device used in an enrichingand isolating methods of the embodiment;

FIG. 4 shows a graph showing changes in concentrations of PCNB, Cl⁻(chloride) and NO₃ ⁻ (nitrate) in a PCNB-containing circulating solutionduring an enriching operation of the present embodiment;

FIG. 5 shows a graph showing differences in enriching speeds ofPCNB-decomposing bacteria in the respective artificial microhabitatsduring an isolating operation of the present embodiment, in relation toconcentrations of chlorine ions (Cl⁻);

FIG. 6 shows a graph showing the states of decomposition of PCNB by sixstrains having a high PCNB-decomposing activity, in relation toconcentrations of PCNB;

FIG. 7 shows a graph showing the states of decomposition of PCNB by thesix strains having a high PCNB-decomposing activity, in relation toconcentrations of chlorine ions (Cl⁻);

FIG. 8 shows a structural formula of CNP;

FIG. 9 shows a graph showing the states of decomposition of CNP by thesix strains having a high PCNB-decomposing activity, in relation toconcentrations of CNP;

FIG. 10 shows a graph showing the states of decomposition of CNP by thesix strains having a high PCNB-decomposing activity, in relation toconcentrations of chlorine ions (Cl⁻); and

FIG. 11 shows a table of results of identification experiment carriedout on three strains having a high PCNB-decomposing activity.

DESCRIPTION OF REFERENCE NUMERALS

1: soil layer tank;

2: enrichment soil layer (enrichment layer);

3: inorganic salt medium; and

4: solution storage tank

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiment of the invention will now be described. The enrichment ofdecomposing bacteria decomposing PCNB by the improved soil percolationtechnique proposed by the present inventors was carried out under thefollowing conditions:

agricultural chemical to be tested: standard product of PCNB (structuralformula thereof is shown in FIG. 1);

soil to be tested: soil of a field repeatedly using PCNB, sieved to soilmaterial having a size of 2 mm or less;

circulating solution: inorganic salt medium containing 10 mg/liter ofPCNB as solo sources of carbon and nitrogen; and

circulating conditions: 25° C., a dark place

As artificial microhabitats for enrichment, out of artificialmicrohabitats A to E having respective physical properties listed inTable of FIG. 2, an artificial microhabitat A was employed. Thefollowing materials A to E in the table are defined as follows:

A: charcoal formed by normally baking a wood of broad-leaved tree at anormal baking temperature of 500° C. (this temperature shall apply for anormal baking temperature hereafter) and fragmenting to pieces of 5 to10 mm;

B: chitosan-treated charcoal formed by normally baking a wood ofbroad-leaved tree and fragmenting to pieces of 5 to 10 mm;

C: charcoal formed by further baking charcoal formed by normally bakinga wood of broad-leaved tree, at 1000° C. for 4 hours, and fragmenting topieces of 5 to 10 mm;

D: charcoal formed by baking conifer wood at 1000° C. for 8 hours andfragmenting to pieces of 5 to 10 mm; and

E: commercially available granular activated carbon (grain diameter of 5mm).

The enrichment of decomposing bacteria according to the embodiment iscarried out in the following manner: As shown in FIG. 3, an enrichmentsoil layer 2 was formed in a soil layer tank 1 having a volume of 500ml. The enrichment soil layer 2 is formed by mixing 2 g of theartificial microhabitat A fragmented to pieces of approximately 5 to 10mm into 40 g of a soil under test. An inorganic salt medium 3 (300 ml)containing PCNB as solo sources of carbon and nitrogen is caused tocirculate from a solution storage tank 4 to the enrichment soil layer 2,for enrichment in a dark place at 25° C.

The results of measurement of concentrations of PCNB, as well asconcentrations of Cl⁻ (chloride) and NO₃ ⁻ (nitrate) as by-products ofdecomposition of PCNB in the circulating solution during the enrichmentoperation are shown in FIG. 4. In FIG. 4, when attention is paid to theconcentration of Cl⁻, it is recognized that after a first replacement ofcirculating solutions (which means that at this time point a circulatingsolution in use is replaced by a new circulating solution newlyprepared; the same applies in the following), the rate of increase inthe concentration of Cl⁻ tends to become higher, and it is confirmedthat after a second replacement of circulating solutions, the rate ofincrease in the concentration of Cl⁻ is drastically increased, meaningthat the enrichment of the decomposing bacteria is in progress. The rateof increase in the concentration of Cl⁻ becomes larger in a mannerproportional to the progress of the enrichment of decomposing bacteria,and means that decomposition of PCNB is being more actively carried outaccordingly.

Then, the artificial microhabitat A having the decomposing bacteriaenriched therein by the above enriching operation was taken out, and0.25 g of the artificial microhabitat A with enrichments and 7.5 g ofeach of new artificial microhabitats A to E are mixed to prepare sixkinds of enrichment layers for isolating the PCNB-decomposing bacteria.The operation for isolating the decomposing bacteria according to theembodiment is carried out by forming the enrichment layers in place ofthe enrichment soil layer 2 shown in FIG. 3 in the soil layer tank 1,and circulating the inorganic salt medium 3 containing PCNB as solosources of carbon and nitrogen through the enrichment layer under thesame conditions as the enriching operation. The results of measurementof concentration of chlorine ions (Cl⁻) as a by-product of decompositionof PCNB when the isolating operation is carried out using the six kindsof artificial microhabitats A to E are shown in FIG. 5. As can beunderstood from the graph of FIG. 5, it is confirmed that only in thecase of the enrichment layer formed by using the artificial microhabitatA, the rate of increase in the concentration of Cl⁻ after a thirdreplacement of circulating solutions becomes higher, and after a fourthreplacement of circulating solutions, the rate of the increase issteeply increased.

Next, the capability of the decomposing bacteria decomposing PCNBobtained by the enriching and isolating operations of the embodimentwill be described. The enriching and isolating operations describedabove are carried out by using the artificial microhabitat A, and 1.0 gof the resulting artificial microhabitat A with enrichments is broken topieces, and properly diluted by using a phosphate buffer solution. Thediluted solution is inoculated into an inorganic salt agar mediumcontaining PCNB as solo sources of carbon and nitrogen, and isolation ofthe decomposing bacteria is carried out by the conventional method, i.e.plate dilution method.

After cultivation for three weeks, clear zones were recognized in aplate medium. Out of the clear zones, one considered to have a highdecomposing activity (large in clear zone area) is used for fishing tenstrains therefrom. The fished ten strains are further cultured in aninorganic agar medium, and then each inoculated in an inorganic saltmedium containing 5 mg/liter of PCNB to examine the decomposing activityof the strains. As a result, six out of ten strains were recognized tohave a high decomposing activity.

The six strains having a high decomposing activity were cultured ininorganic salt agar media, and then a loopful—using an inoculatingneedle—of each strain was inoculated to 30 ml of an inorganic saltsolution containing 3.66 mg/liter of PCNB to examine the decomposingactivities. The results are shown in FIGS. 6 and 7. It was recognizedthat the six strains having a high decomposing activity (PD1, PD3, PD4,PD6, PD9, PD10) each completely decomposed PCNB in five days. Further,FIG. 7 shows results of measurement of chlorine ions (Cl⁻) as aby-product of decomposition of PCNB, whereby it was confirmed that thesix strains each increased the concentration of Cl⁻ to approximately 2mg/liter in five days. This value is substantially equal to 2.20mg/liter i.e. a theoretical Cl⁻ concentration value which is to bemeasured when 3.66 mg/liter of PCNB is completely decomposed, and showsthat the six strains enriched and isolated by the methods according tothe present invention are capable of removing all chlorine atoms in eachPCNB molecule (see FIG. 1) therefrom.

The six strains having a high PCNB-decomposing activity are used toevaluate their capability of decomposing CNP which is mentioned as oneof organochlorine agricultural chemicals difficult to decompose (thestructural formula of CNP is shown in FIG. 8). FIGS. 9 and 10 showresults of experiments carried out by inoculating a loopful—by using aninoculating needle—of each of the six strains to 30 ml of an inorganicsalt solution containing 4.26 mg/liter of CNP for measurement of thePCN-decomposing activity. As shown in the graph of FIG. 9, in all of thesix strains having a high PCNB-decomposing activity, the CNPconcentration of the inorganic salt solution was decreased from 4.26mg/liter to approximately 1 mg/liter in four days, whereby it could beconfirmed that CNP was being decomposed. However, the decomposition doesnot proceed further even after the lapse of four days. Further, fromresults of measurement of concentration of chlorine ions (Cl⁻) as aby-product of CNP decomposition shown in the graph of FIG. 10, evendecomposing bacteria PD1 presumably having a highest decomposingcapability is recognized to have increased the Cl⁻ concentration to onlyapproximately 0.27 mg/liter at the maximum. Considering that atheoretical chlorine ion concentration to be attained when 4.26 mg/literof CNP is completely decomposed is equal to 1.42 mg/liter, thedecomposition of CNP by the six strains can only remove part of threechlorine atoms present in each CNP molecule the structural formula ofwhich is shown in FIG. 8.

Next, the identification of decomposing bacteria carried out by usingthe six strains having a high PCNB-decomposing activity (PD1, PD3, PD4,PD6, PD9, and PD10) will be described. By using inorganic salt agarmedia containing PCNB as solo sources of carbon and nitrogen,purification of the six strains is repeatedly carried out according to aconventional method. Out of the purified six strains, three strainshaving an even higher PCNB-decomposing activity are selected (PD1, PD3and PD6). As to the selected three strains, observation of forms ofbodies of the decomposing bacteria (in respect of Gram staining, shapes,motility, and shapes of colonies), analysis of aliphatic acid of bodiesof the bacteria, quinone analysis, and DNA base composition analysis(content of G+C) were carried out for identification of the decomposingbacteria.

Results of the above experiment for identification of the three strainsare shown in Table of FIG. 11. All of the three strains showed that theyhave a single rod shape, no motility, and negativity of Gram staining.Further, all the three strains can be cultured on bouillon agar plateand form yellow colonies. Then, from the results of the aboveobservation of forms as well as analysis of aliphatic acid of bodies ofthe bacteria, quinone analysis, and DNA base composition analysis, thesethree strains were identified to be bacteria having a scientific name ofBurkholderia cepacia (Burkholderia cepacia KTYY97, received on May 18,1998 by National Institute of Bioscience and Human-Technology, Postalcode: 305-0046, No. 1-1-3, Higashi, Tsukuba City, Ibaraki Prefecture,Japan, Receipt No. FERM BP-6721) [identification is carried out based onthe following documents: “International journal of SystematicBacteriology”, Vol. 47, No. 4, 1188-1200, (1997), the same journal, Vol.45, No. 2, 274-289, (1995), “Handbook of New Bacterial Systematics”,196-231, Academic Press, (1993), “Bacterial Identification Methodaccompanying New Systematics” compiled by Education Committee ofJapanese Society for Bacteriology, Saikon Shuppan, 56-64 (1987).]Burkholderia cepacia is aerobic bacteria which have a high capability ofdecomposing an organochlorine agricultural chemical PCNB as descriedabove.

INDUSTRIAL APPLICABILITY

As described heretofore, the decomposing bacteria Burkholderia cepaciaof the present invention are capable of decomposing an organochlorineagricultural chemical PCNB, and further one of organochlorineagricultural chemicals difficult to decompose, CNP. This decomposingbacteria are aerobic bacteria, which can be easily applied to dispose ofagricultural chemicals remaining in soil, and contributes to a techniqueof preventing contamination of river water and groundwater byagricultural chemicals.

What is claimed is:
 1. A biologically pore culture of aerobic bacteriaBurkholderia cepacia KTYY97 which aerobically decompose anorganochlorine agricultural chemical pentachloronitrobenzene (PCNB). 2.A microorganism according to claim 1, wherein when about 30 ml of aninorganic salt solution containing about 3.66 mg/liter PCNB isinoculated with said microorganism, the PCNB is substantially decomposedwithin about five days.
 3. A microorganism according to claim 2, whereinthe concentration of Cl− in said solution increases to about 2 mg/literwithin about five days.
 4. A microorganism according to claim 1, furthercapable of reducing the chlornitrofen (CNP) concentration of about 30 mlsolution of inorganic salt from about 4.26 mg/liter to about 1 mg/literwithin about four days.
 5. A method of enriching PCNB-decomposingaerobic bacteria Burkholderia cepacia KTYY97 in a Fragmented porousmaterial comprising mixing a soil containing said aerobic bacteria andPCNB with a fragmented porous material having a number of micropores andat the same time a greater adsorptivity for adsorbing PCNB than saidsoil to form an enrichment soil layer, and circulating through saidenrichment soil layer an inorganic salt medium containing a carbon andnitrogen source, said carbon and nitrogen source being formed only ofPCNB, thereby enriching said fragmented porous material with saidaerobic bacteria Burkholderia cepacia.
 6. A method of preparingdecomposing bacteria Burkholderia cepacia KTYY97 for isolation thereofcomprising mixing said fragmented porous material enriched with saidaerobic bacteria Burkholderia cepacia prepared by the method of claim 5into a new fragmented porous material to form an enrichment layerconsisting of the fragmented porous material only, and circulatingthrough said enrichment layer an inorganic salt medium containing acarbon and nitrogen source, said carbon and nitrogen source being formedonly of PCNB, thereby effecting increasing said aerobic bacteriaBurkholderia cepacia in said new fragmented porous material.
 7. Anorganochlorine agricultural chemical-decomposing holdback carriercomprising a porous material holding aerobic bacteria Burkholderiacepacia KTYY97 obtained by the method of claim
 6. 8. An organochlorineagricultural chemical-decomposing holdback carrier comprising a porousmaterial holding PCNB-decomposing aerobic bacteria Burkholderia cepaciaKTYY97.
 9. A carrier according to claim 8, wherein said porous materialholding aerobic bacteria is obtained by: mixing a soil containingaerobic bacteria Burkholderia cepacia KTTY97 and PCNB with a fragmentedporous material having a number of micropores and at the same time agreater adsorptivity for adsorbing PCNB than said soil to form anenrichment soil layer; and circulating an inorganic salt mediumcomprising a carbon and nitrogen source through said enrichment soillayer, said carbon and nitrogen source being formed only of PCNB to formsaid porous material.
 10. A carrier according to claim 8, wherein saidporous material holding aerobic bacteria is obtained by: mixing a firstporous material enriched with said aerobic PCNB-decomposing bacteriawith a second porous material to form an enrichment layer; and,circulating an inorganic salt medium containing a carbon and nitrogensource through said enrichment soil layer, said carbon and nitrogensource being formed only of PCNB to form a further enriched porousmaterial.
 11. A method of enriching PCNB-decomposing aerobic bacteriaBurkholderia cepacia KTYY97 in a porous material, comprising: mixing asoil comprising said anaerobic bacteria and PCNB with a first porousmaterial to form an enrichment layer, wherein the adsorptivity foradsorbing PCNB of said porous material is greater than the adsorptivityfor adsorbing PCNB of said soil; and circulating an inorganic saltmedium comprising a carbon and nitrogen source through said enrichmentlayer, said carbon and nitrogen source being formed only of PCNB toproduce a first enriched porous material.
 12. A method according toclaim 11, wherein said porous material is up to about ten mm is size.13. A method according to claim 11, wherein said porous material is acharcoal material.
 14. A method according to claim 11, furthercomprising: mixing the enriched first porous material with a secondporous material to form an enrichment layer; and circulating aninorganic salt medium containing a carbon and nitrogen source throughsaid enrichment layer; said carbon and nitrogen source being formed onlyby PCNB to produce a further enriched porous material.
 15. A method ofisolating decomposing PCBNB-decomposing bacteria Burkholderia cepaciaKTYY97 by means of decomposing bacteria Burkholderia cepacia KTYY97 bymeans of the enriching method as set forth in claim 14, furthercomprising isolating said bacteria from the enriched first and secondporous materials.
 16. A method according to claim 14, wherein said firstand second porous materials are charcoal materials.